Na, K-ATPase, which is also called the sodium pump, is a membrane-embedded protein found in all animal cells. Despite the important role of Na,K-ATPase in animal cell physiology, the mechanism of ATP-coupled ion transport catalyzed by this protein is unknown. This application describes several experiments to obtain structural data about the ATP binding site(s), the ion selectivity filter, and the interface between A and B subunits of Na,K-ATPase. Isotope-edited NMR measurements will be used to determine the identity of amino acids in proximity to CrATP, Mn2+, or V043- bound to heterologously expressed soluble domains of Na,K-ATPase that are folded in an E2-like conformation. The structure of one of these domains, corresponding to the nucleotide-binding domain N in the crystal structure of Ca-ATPase, will be determined at high-resolution using multidimensional NMR. A new hypothesis to explain the mechanism of ion selectivity by P-type ion pumps is proposed and experiments will be done to test predictions of this hypothesis. Both site-directed mutagenesis and random mutagenesis will be used in biological screens of Na,K-ATPase expressed in yeast to identify amino acids that mediate interactions between Na,K-ATPase and the transported cations. The hypothesis that the cytoplasmic loop between transmembrane segments TM6 and TM7 of the A subunit is involved in the initial binding of cations will be tested by site-directed mutagenesis and chimera formation. Na,K-ATPase and the H,K-ATPases are the only members of the family of P-type ATPases that require two subunits (A and B) for function. In this project, a structural and mechanistic model to explain the role of B in pump function is proposed, and experiments will be done to test certain predictions of the model. In many of the experiments described in the application, the wild type Na,K-ATPase, mutant Na,K-ATPase molecules, and the chimeric pumps Will be expressed in yeast cells because yeast do not contain endogenous Na,K-ATPase and measurements can be made in the absence of an endogenous Na,K-ATPase background activity. Results should permit development of a high-resolution model for binding and hydrolysis of ATP by Na,K-ATPase, provide new insight into the mechanism of ion transport by P type ion pumps, and define locations of important subunit contacts within the protein structure.